The Ser/Thr Kinase MAP4K4 Controls Pro-Metastatic Cell Functions

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Review Article Open Access The Ser/Thr Kinase MAP4K4 Controls Pro-Metastatic Cell Functions Dimitra Tripolitsioti1, Michael A Grotzer1,2 and Martin Baumgartner1* 1Department of Oncology, Children’s Research Center, University Children's Hospital Zürich, August-Forel Strasse 1, CH-8008 Zürich, Switzerland 2Department of Oncology, University Children’s Hospital Zürich, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland *Corresponding author: Martin Baumgartner, Department of Oncology, University Children's Hospital Zürich, Children’s Research Center, August-Forel Strasse 1, CH-8008 Zürich, Switzerland, Tel: +41 44 634 88 20; Fax: +41 44 634 88 59; E-mail: [email protected] Received date: December 20, 2016; Accepted date: January 31, 2017; Published date: February 03, 2017 Copyright: © 2017 Tripolitsioti D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

The search for novel targeted therapies for major human conditions such as diabetes, cardiovascular diseases and cancer is a slow and costly process. Progress is often hampered by poor drug efficaciousness in the patients, low selectivity/specificity of the compounds and cellular evasion mechanism that are rather common in anti-cancer therapies. This is particularly true also for compounds inhibiting kinases, which in theory are optimal targets thanks to their druggable enzymatic activity. Novel targeting strategies are needed to reduce side effects and treatment failure caused by non-specific drug function and target resistance, respectively. An ideal compound will repress the relevant kinase effector function, while leaving kinase functions that are not disease-relevant unaltered. To achieve function-specific inhibition, the molecular mechanism of the drug target that governs the pathological process, must be identified.

The Ser/Thr kinase MAP4K4 is implicated in inflammatory and metabolic disorders and cancer progression. In this review, we describe the molecular effector functions of MAP4K4 that exert those activities and how they have been identified and characterized both in invertebrate organisms and mammals. We discuss how the modulation of the cellular cytoskeleton by MAP4K4 may be connected to pathological conditions such as aberrant angiogenesis and cancer metastasis, and we describe the molecular mechanisms that are so far known to be mechanistically involved in these processes.

Keywords: MAP4K4; Cytoskeleton; Cancer metastasis; Cancer only a few interactors have so far been described, highlighting the still progression incomplete understanding we currently have of how MAP4K4 exerts its functions. Most of the so far identified MAP4K4 interactors are Introduction functionally associated with the modulation of the actin and microtubule cytoskeletons. It is thus tempting to speculate that MAP4K4 (Mitogen activated protein kinase kinase kinase kinase 4, MAP4K4 modulates cell behavior via targeting the underlying also known as Hepatocyte progenitor kinase-like/germinal center cytoskeleton dynamics. In this review, we will particularly focus on the kinase-like kinase (HGK) and Nck-interacting kinase (NIK)) is a role of MAP4K4 in cancer and cancer progression and discuss how serine/threonine protein kinase and member of the human STE20 MAP4K4-controlled cytoskeleton dynamics could initiate and sustain family kinases; it was first identified as a kinase interactor of the the complex morphodynamic processes associated with cancer adaptor protein Nck in mice [1] and short time later the human initiation and progression. orthologue was cloned as well [2]. The MAP4K4 gene is located on chromosome 2 at position q11.2 and encodes from 33 exons a protein Signaling Through MAP4K4 containing 1288 amino acids. Structurally, MAP4K4 contains an N- terminal kinase domain, a coiled-coil domain, a C-terminal Since its discovery in the late nineties, only relatively few details on hydrophobic leucine-rich citron homology domain (CNH) and two the regulation of MAP4K4 activity and its substrates could be revealed. putative caspase cleavage sites [3,4]. An interdomain connecting the This is all the more surprising, considering the wealth of its kinase and the CNH domain is involved in protein-protein interactions implications in development, metabolic disorders and cancer. but is structurally and functionally only poorly understood. Alternative Convincing evidence in different mammalian and fly cell systems splicing results in five isoforms that display differences in interdomain revealed MAP4K4 functions closely associated with the tumor necrosis composition, while kinase and CNH domain are 100% homologous. factor alpha (TNFα)-induced c-jun N-terminal kinase (JNK) signaling MAP4K4 is well conserved and orthologues are found across species. pathway [1,2,5,6]. TNFα-induced JNK activation by MAP4K4 involves Genetic interference and gene expression analyses have implicated signal transmission through the kinases TAK1, MKK4 and MKK7 [2]. MAP4K4 activity in a plethora of cellular functions relevant for Interestingly, MAP4K4 not only mediates TNF-α signaling but also physiological and pathophysiological processes, including organ promotes TNFα expression [2], while TNFα can also cause the specific development, systemic inflammation, metabolic disorders and cancer. increase in MAP4K4 expression via the transcription factors c-Jun and ATF2 [7,8]. On the one hand, this positive feed-back regulation of The astounding bandwidth of these functions, which are further TNFα signaling through MAP4K4 is likely involved in physiological detailed below, indicates the interaction of MAP4K4 with a range of cell functions during development and for tissue homeostasis. On the structurally and functionally different effectors. Surprisingly however,

J Carcinog Mutagen, an open access journal Volume 8 • Issue 1 • 1000284 ISSN:2157-2518 Citation: Tripolitsioti D, Grotzer MA, Baumgartner M (2017) The Ser/Thr Kinase MAP4K4 Controls Pro-Metastatic Cell Functions. J Carcinog Mutagen 8: 284. doi:10.4172/2157-2518.1000284

Page 2 of 8 other hand, aberrant TNFα signaling through MAP4K4 was also linked to pathophysiological processes using genetic ablation and pharmacological inhibition approaches. These processes include insulin sensitivity [8], systemic inflammation [9], pathogen-dependent oncogenic progression [10,11] and vascular inflammation and atherosclerosis [12]. In addition to the TNFα-receptor, growth factors (GF) such as PDGF or EGF activate MAP4K4 through receptor tyrosine kinases (RTKs) [2] and trigger the phosphorylation and activation of the MAP4K4 substrates sodium- proton exchanger [1] (NHE1) [13], ezrin, radixin, moesin (ERM) family proteins [14] and actin-related protein [2] (Arp2) [15]. Genetic studies in C. elegans identified an interaction between the worm ortholog of MAP4K4–MIG-15–and integrin/PAT3 [16]. Whether integrin receptor engagement could activate MAP4K4 signaling analogous to the TNFα receptor or the RTKs described above is currently not known. However, compelling evidence indicates that MAP4K4 decreases surface availability of active integrins [17], for example by inactivating integrins via moesin phosphorylation, which concomitantly competes with talin for integrin binding [18], or by Figure 1: Overview of the major MAP4K4 signaling pathways. accelerating their endosomal trafficking [19]. Still in the context of integrin signaling, MAP4K4 was found to be phosphorylated by the focal adhesion kinase Pyk2 [20], which is From Insulin Resistance to Cell Motility: The Panoply regulated by intracellular calcium levels and activated by G-protein- of MAP4K4 Control of Physiological Processes coupled receptors [21]. Although not directly activated by transforming growth factor ß/ Glucose up-take and insulin function bone morphogenic protein (TGFß/BMP), MAP4K4 as well as the The pathological failure of cells to respond to insulin with glucose closely-related TNIK and MINK promote inhibitory phosphorylation up-take and metabolization is referred to as insulin resistance. It causes of Thr312 in the TGFß/BMP effector R-Smads1/2/3/5/8 [22]. blood glucose levels to raise above the normal range, is associated with Somewhat counterintuitively, MAP4K4 along with its relatives obesity, hypertension and cardiovascular diseases and eventually MAP4K1-6, were recently also found to phosphorylate and activate the results in type 2 diabetes [25,26]. Causative mechanisms for insulin kinase Large Tumor Suppressor 1/2 (LATS1/2) under conditions of resistance include impaired insulin receptor (IR) signaling and the serum starvation [23], which inactivates Yes-associated protein 1 concomitantly deficient ability to restrict hepatic glucose output, as (YAP1) in the evolutionary conserved Hippo pathway. Hippo pathway well as reduced uptake of glucose into muscle and adipose tissue activation and phosphorylation-dependent cytosolic retention of the through the glucose transporter GLUT4. An siRNA screen identified YAP transcriptional regulator is tumor suppressive, as it prevents pro- MAP4K4 as a negative regulator of GLUT4 control of glucose proliferative and anti-apoptotic YAP signaling [24]. metabolism in adipocytes that impairs GLUT4 expression and function 8. Inflammation-associated TNFα regulates insulin action Together, a still incomplete picture begun to emerge, where negatively by repressing signaling downstream of the insulin receptor MAP4K4 activation in membrane proximal, upstream compartments and by preventing GLUT4 translocation. Depletion of MAP4K4 occurs in response to TNFα receptor, RTKs, and–possibly–also G- ameliorates systemic inflammation in a murine model by reducing protein-coupled receptor ligation, to trigger JNK and Hippo pathway TNFα expression in macrophages [9]. This led to the hypothesis that activation, sodium-proton exchange, actin polymerization and plasma MAP4K4 could also mediate the detrimental effects in glucose membrane remodeling (Figure 1). metabolizing cells exposed to systemic TNFα. Indeed, silencing MAP4K4 reverted TNFα-dependent IRS2 depletion and insulin repression in beta cells [27], and it restored peroxisome proliferator- activated receptor gamma (PPARgamma) expression by de-repression of mTORC for a normalized glucose and lipid metabolism in adipocytes [28]. Consistently, microRNA 30d repression of MAP4K4 expression in pancreatic cells protected beta-cells against TNFα- induced suppression of both insulin transcription and secretion, further confirming MAP4K4 targeting as a beneficial strategy for diabetes prevention [29]. MAP4K4 has also been suggested as a potential biomarker in non- obese type 2 diabetes, since decreased MAP4K4 expression rates- attributed to increased methylation of the MAP4K4 promoter-were observed in non-obese type 2 diabetes patients compared to healthy subjects [30]. Further in vivo studies demonstrated that MAP4K4 deletion led to decreased fasting blood glucose concentrations and

Page 3 of 8 enhanced insulin signaling in adipose tissue and liver [31], and that phosphorylation of the Hippo pathway core component LATS1/2 and MAP4K4 mediates hyperinsulinemia in chronic obesity by promoting concomitant degradation of the transcriptional regulators YAP/TAZ islet hypertrophy and insulin secretion from pancreatic cells [32]. [41]. MAP4K4 is also implicated in vascular inflammation and the formation of atherosclerotic plaques, where it enhances TNFα-induced Axon Navigation and Neurite Outgrowth vascular endothelial permeability, macrophage recruitment and adhesion [12]. Axonal migration during development is a complex process that controls the correct orientation and connection of axons. MAP4K4 is These clear indications of the functional significance of MAP4K4 in involved in axonal navigation and neuronal pathfinding with the first inflammation-associated pathologies call for the use of therapeutics evidence being identified in Drosophila with the identification of Msn/ specifically targeting MAP4K4. However, the discovery of conditional MAP4K4 control of photoreceptor pathfinding [42]. Shortly after, the MAP4K4 deficiency in T cells causing systemic inflammation and genetic interaction of MIG-15/MAP4K4 and the integrin Ina-1 was insulin resistance in mice sound a note of caution [33]. In T cells, found to be required for proper axon navigation in C. elegans [16]. MAP4K4 directly phosphorylates TNF receptor-associated factor 2 While MIG-15 appears to be dispensable for lamellipodia and (TRAF2), leading to its degradation, repression of IL-6 production and filopodia dynamics during growth cone outgrowth and cell migration, the concomitant suppression of Th17 cell differentiation, which it is required for proper directional control of neuronal outgrowth [43]. triggers an insulin resistant phenotype. Another study revealed that Comparative time lapse imaging of growth cone dynamics revealed MAP4K4 expression in CD4+ T-cells restricts lung inflammation in that MIG-15 mechanistically interacts with the ezrin/radixin/moesin response to the environmental pollutant ozone-oxidized black carbon ortholog ERM-1 at the rear end of the growth cone, to restrict lateral (oBC), which is characterized by increased numbers of macrophages, protrusions and to maintain directional migration [44]. In mammalian lymphocytes, neutrophils, cytokine secretion and bronchoalveolar cells, MAP4K4 was identified in a high-throughput screen for lavage fluid accumulation (BALF) [34]. Finally, mice lacking regulators of neurite outgrowth. In contrast to its neuro-promoting endothelial MAP4K4 displayed lymphatic defects such as dilated functions during development, MAP4K4 depletion in cerebellar lymphatic capillaries, insufficient lymphatic valves, and impaired granule neurons significantly increased the length of regenerating lymphatic flow [35], suggesting that beneficial effects of MAP4K4 neurites, suggesting that MAP4K4 in mammals restricts neuronal targeting in the atherosclerotic blood vasculature may have negative outgrowth [45]. This finding was confirmed by an unrelated study impact on lymphatic drainage. Thus, careful future studies will be using a militarinone-inspired 4-hydroxy-2-pyridone inhibitor targeting required to assess the cell-type specific functions of MAP4K4 and their MAP4K4, which resulted in neuritogenesis [46]. MAP4K4 inhibition is role in metabolic and inflammatory diseases. also a reported target of kenpaullone, an inhibitor of Amyotrophic Lateral Sclerosis (ALS) [47]. Consistent with a potentially destructive Development and Differentiation function of MAP4K4 for neuronal integrity, kenpaullone improved the survival of human motor neurons derived either from mouse The Drosophila ortholog of MAP4K4 – misshapen (MSN) – was embryonic stem cells or from ALS-patient-induced pluripotent stem initially identified as shape control gene in the Drosophila eye [36]. cells. Interestingly, kenpaullone efficiency was partially attributed to its Subsequent studies implicated it upstream of Drosophila JNK to inhibition of the MAP4K4-TAK1-MKK4-JNK-c-Jun cell death control dorsal closure during late embryogenesis [5]. In mammals, signaling cascade. MAP4K4 plays a critical role in stimulating migration of presomitic mesodermal cells away from the Primitive Streak (PS) and in Together, these somewhat conflicting results from developing promoting the differentiation of presomitic mesoderm into somites organisms and mature neurons corroborate the relevance of proper [37]. Its absence causes embryonic lethality between days 9.5 and 10.5. MAP4K4 function for neuronal development and integrity. However, MAP4K4 is also involved in the control of epithelial to mesenchymal future research will be necessary to reconcile the stabilizing functions transition (EMT) during gastrulation, where it along with the p38- of MAP4K4 during neuronal development with its destructive interacting protein (p38IP) activates p38 to downregulated E-cadherin signaling in mature neurons. It will also be of particular interest to expression [38]. E-cadherin down-regulation contributes to the explore whether kenpaullone could be used to treat brain cancers with mesenchymal phenotype, which is prerequisite for cells to move away increased MAP4K4 expression such as glioblastoma [3,48] and from the primitive streak. Interestingly, the above signaling cascade medulloblastoma [49]. functions independently from the well characterized EMT-associated pathway that involves FGF and the Snail transcription factor, and MAP4K4 is Overexpressed in Solid Tumors suggests an alternative route requiring MAP4K4 activation of p38 [38]. A comparative analysis of healthy tissues with lines of the National TGFß/BMP signaling, which is involved in early development, axis Cancer Institute tumor panel reveled striking up-regulation of formation and patterning [39], is negatively regulated by MAP4K4 and MAP4K4 in latter, with glioblastoma cancer cell lines displaying the prevented from exerting its correct function during Drosophila highest upregulation in MAP4K4 expression levels [3]. Since then, a imaginal disk formation [22]. Although speculative, MAP4K4 could number of studies listed below have noted increased expression and/or thus be involved in spatio-temporal fine tuning of TGFß/BMP altered function of MAP4K4 in tumors. signaling during development. It is also conceivable, that alterations in this regulation in the context of aberrant MAP4K4 activation could Brain tumors result in developmental defects in the embryo and cancer initiation/ progression in the adult organism. In addition to its role in the Transcription analyses in glioblastoma multiforme cell lines and developing organ, MAP4K4 also contributes to reshaping the adult primary samples revealed that EGFRvIII, a constitutively activated organism, where it restricts differentiation of myoblasts into myotubes EGFR mutant, leads to specific upregulation of a group of genes [40] or in enteroblasts, where it controls differentiation by including MAP4K4 [48]. Also in a glioblastoma model, MAP4K4 was

Page 4 of 8 found to interact with the focal adhesion-related kinase Pyk2 and to 3’-untranslated region (3’-UTR) of target messenger RNAs [57]. cooperate with it for glioblastoma cell migration [20]. MAP4K4 Several MAP4K4-targeting miRNAs were found repressed in tumors, expression is also markedly higher in pediatric Medulloblastoma, leading thus to increased levels of MAP4K4 expression. These include particularly in the SHH and Group 4 subgroups, compared to healthy Let7a in Kaposi sarcoma, miR-194 in CRC [58] and HCC [59] and cerebellum tissue, and promotes tumor cell migration and invasion miR-622 in CRC [60]. downstream of the hepatocyte growth factor receptor c-Met [49]. MAP4K4 Controls Pro-Metastatic Functions in Cancer Pancreatic ductal adenocarcinoma Progression MAP4K4 is overexpressed in 46% of stage II Pancreatic Ductal Increased expression levels of MAP4K4 at mRNA or protein levels Adenocarcinoma (PDA) primary samples, with particular have been correlated with tumor aggressiveness and reduced patient overexpression detected in the neoplastic epithelium compared to the survival. But what are the molecular events controlled by MAP4K4 PDA stroma [50]. Furthermore, multivariate statistical analyses in that trigger or maintain the transformed cellular phenotype and PDA samples revealed the association of MAP4K4 expression with promote metastatic disease? Despite research efforts ongoing for close poor overall and recurrence-free survival, recommending it as a to 20 years, this question still remains incompletely answered. prognostic marker for patients with stage II PDA [51]. Improved gene interference strategies and the development of small compound inhibitors of MAP4K4 have simplified the identification of Colorectal cancer effector pathways and putative molecular interactors. Nevertheless, the MAP4K4 and four additional genes (LYN, SDCBP, DKK1, and effector molecules of MAP4K4 and how their interaction with altered MID1) consist a five-gene signature in colorectal cancer highly MAP4K4 expression or function is coupled to the biological outcome, correlated with poor overall survival of the patients. In addition, remains enigmatic in most instances. Key biological functions increased MAP4K4 mRNA expression per se closely correlates with controlled by MAP4K4 are reviewed in the following section. metastasis, tumor invasion and decreased overall patient survival [52]. Tumor cell growth Lung adenocarcinoma Ras-transformation of NIH3T3 fibroblasts enables these cells to Adenocarcinoma is the most common histological subtype of lung grow independent of adhesion to a rigid matrix (anchorage cancer and accounts for almost half of all lung cancers. Elevated independent growth). Ablation of MAP4K4 function in such NIH3T3 MAP4K4 expression is closely associated with lung adenocarcinoma fibroblasts by the overexpression of a kinase-inactive MAP4K4 mutant progression and is an independent prognostic factor predicting poor completely abrogated anchorage independence, suggesting that overall survival [53]. MAP4K4 contributes an essential function to anchorage-independent survival and growth [3]. Knock-down of MAP4K4 caused cell cycle Hepatocellular carcinoma (HCC) arrest and apoptosis in HCCs [54] and in gastric cancer cells (GCCs) [55]. Cell cycle arrest and apoptosis after MAP4K4 knock-down was MAP4K4 expression is increased in Hepatocellular carcinoma accompanied by the repression of JNK, NF kappa B, and toll-like (HCC) specimens compared to adjacent non-tumor liver tissues, and receptor (TLR) signaling in HCC cells [54], and increased the Bax/ its upregulation correlates positively with tumor size, tumor grade and Bcl-2 ratio and decreased Notch signaling in GCCs [55]. Whether intrahepatic metastasis. Consistently, high MAP4K4 expression is an impaired HCC and GCC growth after MAP4K4 ablation is indeed the independent predictor of poor prognosis in HCC patients [54]. consequence of repressed JNK, NF-kappa B and TLR signaling, or rather mediated by an alternative route in these cancer cell lines is not Gastric cancer clear. However, since MAP4K4 ablation does not cause growth arrest and apoptosis in all cancer cell lines, the susceptibility factor in the Gastric cancer is the second most common cause of cancer- sensitive ones remains to be determined. associated mortality worldwide. In more than 2/3 of gastric cancer patient samples increased MAP4K4 expression levels were detected compared to adjacent non-tumorous regions [55]. Cell migration and invasion Early studies in Drosophila and C. elegans pointed towards cell Ovarian cancer shape and migration control as hallmark functions of MAP4K4 signaling [5,36,61]. Since disturbed morphodynamics and cell In contrast to all other solid tumors investigated, MAP4K4 migration control are intimately associated with the cancerous expression levels correlate negatively with tumor load in a murine phenotype, studies in cancer cell lines soon confirmed the pro- model of ovarian cancer. In this study, non-transformed ovarian migratory function of MAP4K4 [3,62]. MAP4K4 implications in epithelial cells were compared to their spontaneously transformed migration control in the context of cell transformation was also shown counterparts using genome-wide transcriptome analysis. Alterations in in macrophages infected with the protozoan parasite Theileria the gene expression profile were then correlated with the profiles of annulata, where parasite-induced TNFα secretion by the host cell four different human ovarian cancer types [56]. activates MAP4K4 in an autocrine manner, to drive migration and invasion of the host cell [11]. In an inflammatory context as well, microRNA regulation of MAP4K4 expression in cancer MAP4K4 promotes Kaposi Herpes virus-infected cell migration by microRNAs (miRNAs) are endogenous, small noncoding RNAs, controlling the expression of genes responsible for motility and with a length of approximately [19–24] nucleotides that negatively invasiveness such as COX-2, MMP-7 and MMP-13. In HCC, MAP4K4 regulate gene expression by base pairing complementary sites on the depletion markedly reduced cancer cell migration both in vitro and in

Page 5 of 8 vivo, reduced cell adhesion to extracellular matrix (ECM) proteins and inside 3D matrix, where focal adhesions, the adhesion associated impaired the expression of NF-κB and the Matrix Metalloproteases 2 signaling complexes and their coordinated turnover controls and 9 [54,63]. In the malignant pediatric brain tumor mesenchymal cancer cell migration [65]. medulloblastoma, MAP4K4 is overexpressed and mediates migration Cytoskeleton dynamics at the cell front during migratoion are and invasion downstream of HGF and EGF [49], whereas in adult controlled among many other factors by local pH gradients. The glioblastoma cells, MAP4K4 was found to interact with Pyk2, a sodium/proton (Na+/H+) exchanger 1 (NHE1) causes spatially- member of the focal adhesion kinase family, and to be required for restricted H+ efflux at the leading edge of cells [66]. In addition to its migration and invasion [20]. Molecular mechanisms linking MAP4K4 exchanger activity, NHE1 also acts as a membrane anchor for the activity to pro-metastatic functions Figure 2. cytoskeleton and an assembly point for signaling molecules [67-69]. MAP4K4 binds to and phosphorylates the C-terminus of NHE1 and causes its activation, which increases the intracellular pH in proximity of the plasma membrane [13]. NHE1 is involved in cell migration control [70-72] and associated with metastatic functions in cancer cells [73-75]. GF control of this central regulator of cortical cell functions through MAP4K4 may contribute to local remodeling of the cytoskeleton [76,77] and enable the formation of invasive membrane protrusions. Not surprisingly, targeting NHE1 has emerged as a potential strategy in tumor treatment [78]. Another possible function of MAP4K4 in cell migration and invasion control is its activity towards the actin-related protein 2 (Arp2) subunit of the Arp2/3 complex. Arp2 phosphorylation by MAP4K4 increased the actin nucleation activity of the Arp2/3 complex in vitro and in EGF-stimulated cells, demonstrating that MAP4K4 can couple growth factor (GF) signaling to F-actin polymerization [15]. Figure 2: Upregulation of F-actin cytoskeleton turnover and MAP4K4 accumulates at the leading edge of cells migrating on/in membrane dynamics at the leading edge of cancer cells facilitate either 2D or 3D matrix [3,11,14,49,79]. Thus, MAP4K4-driven F-actin migration and tissue invasion. Small compound inhibitors targeting polymerization at the leading edge combined with its activity towards MAP4K4 block these pro-migratory functions and cells revert to a focal adhesions make it a prime candidate for GF control of the more stationary and less migratory phenotype. membrane protrusions at the leading edge of cells that drive invasive cancer cell motility. Indeed, depletion of MAP4K4 in medulloblastoma cells infiltrating collagen I matrix caused a defect in protrusion Mechanistically, the identification of C. elegans integrin as an formation and completely abrogated GF-induced cancer cell invasion interaction partner of MAP4K4 during commissural axon navigation [49]. [16] hinted towards functional implication of MAP4K4 in cell adhesion and migration control. The ezrin, radixin, moesin ERM Therapeutics Targeting MAP4K4 Functions family were then identified as MAP4K4 substrates and interactors, which mediate lamellipodium extension in response to growth factor The broad implication of MAP4K4 in human disease conditions (GF) stimulation in breast adenocarcinoma cells [14] and in highly including metabolic and inflammatory disorders as well as cancer have motile macrophages transformed by intracellular pathogen Theileria incited efforts in the pharmaceutical industry to develop a specific annulata [11]. Interestingly, in C. elegans, MIG-15 and the moesin small compound inhibitor. Genentech applied two separate fragment- orthologue ERM-1 function in the same pathway to direct based lead discovery approaches, which identified pyridopyrimidine commissural axon migration [44]. Mechanistically, MIG-15 and scaffold-based compound 29 (C29) [80] and C35 [40]. Starting from a ERM-1 enable the stabilization of the rear end of the growth cone and micromolar pyrrolotriazine fragment hit, Genentech developed a series the retraction of non-productive protrusions. Whether this structural of MAP4K4 inhibitors with single digit nanomolar IC50s by utilizing stabilization of the growth cone by MIG-15 and ERM-1 in C. elegans is crystal structures and molecular modeling. Calculated properties were mechanistically related to lamellipodium stabilization in mammalian used to guide the optimization of solubility and in vitro stability, cells is not clear. Consistent with the notion that MAP4K4 controls cell yielding compounds with excellent biochemical potencies, ligand adhesion are two recent studies describing accelerated turnover of efficiencies and favorable in vivo bioavailability in mice. However, as integrin-based adhesions: On the one hand Yue et al. identified with many small compound kinase inhibitors, moderate kinase MAP4K4 as a focal adhesion (FA) disassembly factor acting in selectivity was observed and the closely related kinase Misshapen-like conjunction with microtubule ending binding protein 2 (EB2) and IQ kinase 1 (MINK1) and TRAF2- and Nck-interacting kinase (TNIK) motif and SEC7 domain-containing protein 1 (IQSEC1) in were also effectively inhibited by both compounds. C35 was found to keratinocytes [64]. On the other hand, Vitorino et al. found that be even less specific as it also blocked activities of Cdk1, Flt3, KDR, endothelial cell MAP4K4 phosphorylates moesin to displace the F- KHS1, and Mink1 by more than 75% [40]. Nevertheless, C29 was actin binding protein talin from the intracellular tail of ß1 integrin found to be a valuable tool in in vivo studies but its applicability [18]. Common to both studies is that MAP4K4 activity triggers the suffered from the shortcoming that it was poorly tolerated when dosed turnover of adhesion complexes. Indeed, depletion of MAP4K4 or its repeatedly [80]. Its capability to permeate into the CSN was suspected inactivation causes the cells to spread and adhere more tightly to cause the poor tolerability. Medical chemistry efforts were therefore [3,11,14]. Acceleration of focal adhesion turnover may thus be one undertaken to reduce penetration of C29 into the CNS. The resulting function of MAP4K4 to facilitate cell displacement, in particularly also GNE-495 reduced brain exposure while maintaining potent activity

Page 6 of 8 and good kinase selectivity, and recapitulated the phenotypes of under the angle of the effects MAP4K4 may have on cytoskeleton MAP4K4 inducible knockout mice [81]. In a search for modulation. An example for such context-dependent, global pharmacologically active compounds targeting pathological modulation of cell function was observed in a host-pathogen model, angiogenesis, the GNE-220–yet another MAP4K4 inhibitor from where parasite-dependent, TNFα-induced MAP4K4 signaling in the Genentech–was identified. This compound prevented MAP4K4- host cells bifurcates into JNK expression and activation on the one dependent talin replacement by C-terminally phosphorylated moesin hand and ERM protein-associated control of lamellipodium dynamics from the intracellular domain of integrin β1, and reversed membrane and invasive motility on the other hand [11]. Through its activity at retraction defects in endothelial cells associated with pathological different levels of cell function, specific targeting of its context- angiogenesis [18]. dependent actions may thus emerge as a therapy specifically tailored to the disease-causing activity, and hence reduce non-desired side-effects A virtual screening approach using the crystal structure of the of treatment. MAP4K4 kinase domain was employed by Pfizer, and the compound PF-6260933 with excellent selectivity was developed [82]. However, similar to the Genentech compounds, PF-6260933 also inactivated Acknowledgments TNIK and MINK. PF-6260933 was used in a study investigating This work was supported by grants of the Swiss National Science MAP4K4 function in vascular inflammation and atherosclerosis. 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